Our long-term goal is to understand the role of telomeres in aging and human disease. Telomeres are the specialized structure at both ends of each of our chromosomes that function to prevent the ends from being treated as broken DNA. If it were not for the capping function of telomeres, chromosomes would fuse together and tear themselves apart during cell division. The DNA replication apparatus cannot copy to the very end of our linear chromosomes, and a special enzyme called telomerase adds DNA to the ends to compensate for the shortening that would otherwise occur. Telomerase is expressed during early embryonic development (which permits telomere length to be maintained and our survival as a species) and in certain of our proliferative stem cells (where it slows but does not prevent telomere shortening from occurring to allow an increased number of cell divisions). Telomerase is turned off in most of our tissues, and the subsequent telomere shortening limits the number of times cells within these tissues can divide. This blocks most pre-malignant cells from acquiring the number of mutations they need to form tumors, but also limits cell turnover and almost certainly contributes to the physiological decline in some tissues associated with aging. Telomeres are much more difficult to study than a typical gene because we have 92 of them (one on each end of our 23 pairs of chromosomes), they are composed many kilobases of repetitive TTAGGG sequences that lack restriction sites, and their size is different for each chromosome end. We have developed a large number of novel techniques to study telomeres that permit us to address many important questions in our field. The present application develops new techniques and extends our current observations in two broad aims. The first aim is to open up an entire new field for the ways by which telomere shortening affects human biology. The field has focused its efforts on the consequences of replicative aging, which occurs when telomeres become so short they produce DNA damage signaling. Our preliminary data demonstrates different mechanisms by which telomere shortening can change the expression of subtelomeric genes throughout life, and one specific disease in which this occurs. We propose to identify many additional genes affected by telomere shortening and their consequences for human aging and disease. Our second aim uses the techniques we have developed to analyze the many processing steps that occur when telomeres replicate, the nature of the structures that prevent the ends from being recognized as damaged DNA, and the nature of one mechanism by which tumor cells escape the barrier of replicative aging. The results of these studies are critical to understanding telomere behavior and for identifying targets for therapeutic interventions to counteract the effects of telomere shortening or treat the mechanisms by which tumor cells prevent telomere shortening.